An atypical form of 60S ribosomal subunit in Diamond-Blackfan anemia linked to RPL17 variants

Diamond-Blackfan anemia syndrome (DBA) is a ribosomopathy associated with loss-of-function variants in more than 20 ribosomal protein (RP) genes. Here, we report the genetic, functional, and biochemical dissection of 2 multigenerational pedigrees with variants in RPL17, a large ribosomal subunit protein–encoding gene. Affected individuals had clinical features and erythroid proliferation defects consistent with DBA. Further, RPL17/uL22 depletion resulted in anemia and micrognathia in zebrafish larvae, and in vivo complementation studies indicated that RPL17 variants were pathogenic. Lymphoblastoid cell lines (LCLs) derived from patients displayed a ribosomal RNA maturation defect reflecting haploinsufficiency of RPL17. The proteins encoded by RPL17 variants were not incorporated into ribosomes, but 10%–20% of 60S ribosomal subunits contained a short form of 5.8S rRNA (5.8SC), a species that is marginal in normal cells. These atypical 60S subunits were actively engaged in translation. Ribosome profiling showed changes of the translational profile, but those are similar to LCLs bearing RPS19 variants. These results link an additional RP gene to DBA. They show that ribosomes can be modified substantially by RPL17 haploinsufficiency but support the paradigm that translation alterations in DBA are primarily related to insufficient ribosome production rather than to changes in ribosome structure or composition.


1-IV-3 (Sibling of the index case).
During the second pregnancy of 1-III-5, prenatal genetic testing was conducted for the RPL17 c.217-3C>G variant.He is not a mutation carrier and is reported to be healthy.

1-III-6 (Maternal uncle)
. Individual 1-III-6 was born at term with bilateral absent thumbs; these were treated surgically by pollicization of the index fingers.He was followed during infancy for a moderate anemia which did not necessitate any treatment.At age 35, his CBC values were within normal limits, except for a low erythrocyte count (4.22 T/L); HGB: 13.5 g/dL; Leukocytes: 4.9 G/L.

1-II-2 (Maternal great-uncle).
This individual presents with retrognathism and a nasal voice.He has been monitored clinically for leukopenia for decades (consistently ~3.0 G/L; Normal: 4-10 G/L), with a major episode when he was 30 years old.His other CBC values were within normal limits at the most recent examination, age 71 yo: HGB: 13.9 g/dL; MCV: 91 fl, total leukocytes 2.7 G/L (mild leukopenia).

1-II-1 (Maternal great-uncle)
He is reported to be healthy and does not carry the RPL17 c.217-3C>G mutation.His most recent CBC values were within normal limits, HGB: 15.4 g/dL; leukocytes: 6.9 G/L; erythrocytes: 4.73 T/L.
Upon physical examination, we noticed a small chin and bilateral 5 th digit clinodactyly.At her last clinic visit at 83 years of age, she displayed leukopenia: 2.5 G/L and moderate thrombocytopenia (140,000-180,000/µl) but no anemia was observed for the past several decades that she was followed by our clinic (erythrocytes: 4.62 T/L ; HGB: 13.8 g/dL).She developed an acute myeloid leukemia and died soon after the diagnosis.She had two sisters and one brother; and three out of four nieces and nephews (children of her deceased brother) had no particular medical history and were tested negative for the RPL17 c.217-3C>G mutation.
1-III-3.She was born at term following an uneventful pregnancy; birth weight: 3050 g (<25 th centile); length: 47 cm (<25 th centile).Severe anemia (HGB: 3.5 g/dL; Normal: 10.5-13.5 g/dL) was discovered when she was 13 months old, and was treated initially by packed red blood cell transfusions and later on by corticosteroids (prednisone: 12 mg/2d).She also presented thrombocytopenia.A diagnostic bone marrow aspirate/biopsy showed severe hypocellularity with marked erythroid lineage hypoplasia and dyserythropoiesis (incl.megaloblastic forms) and mild dysmyelopoiesis.The pattern was deemed to be compatible with DBA.After introduction of prednisone therapy, repeat bone marrow aspirates/biopsies showed progressive correction of the previously observed features without complete correction of the hypocellularity.Prednisone treatment was diminished progressively (5 mg/2 days at age 3.5 years old).When she was 4 years 6 months, steroid treatment was stopped; her HGB and platelet levels remained stable, but leukoneutropenia was observed.Upon physical examination, she displays a small chin, and fifth finger clinodactyly.At age 32, a hematological evaluation confirmed the diagnostic of a minor functional platelet disorder.There was no anemia, HGB: 14.3 g/L; Erythrocytes: 4.55 T/L; MCV: 94 fl.However, mild leukopenia was observed; leucocytes: 3.0 G/L without neutropenia.Followup leukocyte levels corrected (3.8-4.3G/L).Her ADA level is in the normal range: 219 U/lEC (N: 100-400).

1-IV-4 (cousin of the index case).
This female is the healthy child of 1-III-6.The parents declined genetic testing of the RPL17 c.217-3C>G mutation.

Family 2 Case report
The index case of Family 2 (2-II-1) is a male delivered at 39 weeks gestation to his primigravid mother by vaginal delivery.Although no pregnancy complications were reported, ultrasound exams were notable for poor fetal growth from early on and he weighed 4 pounds 10 ounces at birth.His neonatal hospital stay was six days due to poor weight gain and hypoglycemia.He was followed by his pediatrician for poor growth.He presented at 8 months of age with acute vomiting and a CBC revealed pancytopenia with macrocytic anemia (HGB of 4.8 g/dL; Normal: 10.5-13.5 g/dL), neutropenia with an absolute neutrophil count (ANC) of 30 G/L (Normal: 1-8 G/L) and mild thrombocytopenia with platelet count of 117 G/L (Normal: 150-450 G/L).A bone marrow aspirate was performed, which showed no evidence of malignancy; testing for EBV, CMV, HIV, and parvovirus was negative.Further workup for congenital bone marrow failure syndromes was recommended but was never sent.He was less than the third percentile for both height (65 cm) and weight (6.5 kg) and was noted to have an incarcerated inguinal hernia which was surgically repaired.At follow up in hematology/oncology clinic at 9 months of age, his counts showed recovery (HGB 9.3 g/dL, WBC 7.85 G/L, platelet 224 G/L) and it was thought that he had recovered from a viral suppression of his marrow.However, he was readmitted to hospital at 10 months of age for fever, pancytopenia and growth failure.Genetics consultation at the time noted mild dysmorphia including a tall forehead, epicanthal folds, a high nasal root with narrow nasal base, full lips with a mildly triangular facial shape.Negative etiologic testing included an infectious disease work-up, cystic fibrosis, vitamin B12/ folate deficiency, karyotype, microarray-CGH, DEB clastogen assay for Fanconi anemia, sequencing for Noonan syndrome (BRAF, HRAS, KRAS, MAP2K1, MAP2K2, PTPN11, RAF1, and SOS1), TIN2, TERC, SBDS, and DKC1.Upon evaluation for Dyskeratosis Congenita, his telomere length on 3 cell lines was short (<1%); repeat detailed testing demonstrated telomere abnormalities in the following cell types: lymphocyteslow; granulocytes-very low; Naive T cells-low; Memory T cells-low; B cells -very low; NK cellslow.These results were reviewed by experts at the National Institutes of Health (NIH) and thought not to be low enough to be diagnostic of Dyskeratosis Congenita.His anemia was initially transfusion dependent, but the last transfusion needed was at 15 months of age.His neutropenia also improved without requiring treatment with granulocyte stimulating factor.His most recent CBC was conducted at 6 years of age: HGB 11.4 g/dL (Normal: 11.5-15.5 g/dL), platelets 143 (Normal: 150-450 G/L) and ANC of 3.3 G/L (Normal: 1.5-9 G/L).His developmental milestones were met on time.He was last seen at the age of 8 and was diagnosed with autism and ADHD; he was following his own growth curve at the 3-5% for both height and weight.His mother and father are 5 '5" and 5'8" tall, respectively; both are healthy, with no history of anemia or developmental problems.A paternal aunt is noted to have a hole in her heart, wide neck and large forehead, resembling the patient's father in both stature and appearance.A paternal uncle is 5 feet 6 inches tall and with a heart murmur.The paternal grandparents are 5'2" and 5'3" tall and healthy.

2-I-1 (Father of the index case).
The father, heterozygous for the RPL17 c.452delC variant, is reportedly healthy.He is 5 feet 8 inches tall.